Anti-inflammatory formulations

ABSTRACT

The invention features compositions and methods for reducing inflammation of ocular tissue by administering to an individual a mixture of ingredients containing carotenoid and a polyphenol compound.

BACKGROUND OF THE INVENTION

The invention relates to control of ocular inflammation.

Aqueous tear-deficient dry eye syndrome is a disruption of the ocularsurface-lacrimal gland homeostatic cycle. It is characterized by dryinflammation of the lacrimal gland, and presence of a dense infiltrateof inflammatory cells in and around the tear duct causing high localizedexpression of pro-inflammatory cytokines. A particularly debilitatingform of the disorder is dry, age-related macular degeneration whichaffects a substantial fraction of the population older than 65 years andis currently incurable

SUMMARY OF THE INVENTION

The invention features an anti-inflammatory composition, which isassociated with reduced adverse side effects compared to conventionalanti-inflammatory drugs. Additionally, the combination of the individualcomponents of the composition results in a greater anti-inflammatoryeffect than the anti-inflammatory effect of the individual componentswhen administered singularly. The anti-inflammatory compositions are areuseful in protecting ocular tissue from inflammation related andoxidative damage.

The anti-inflammatory composition contains a carotenoid and apolyphenol. The carotenoid is mixed carotenoid compound, an astaxanthinor a zeaxanthin. The polyphenol is curcuma longa root powder, green tea,grape seed extract, cinnamon, or a citrus bioflavonoid. The cinnamon isin the form of ground whole cinnamon bark or leaves, extracted cinnamonoil from bark or leaves, or a water-soluble cinnamon extract. Forexample, the cinnamon bioflavonoid is a water-soluble type A polyphenolsuch as a methyl hydroxy chalcone polymer (MHCP). Alternatively, thepolyphenol is a cox-2 inhibitor such as a quercetin, a bilberry extract,a hops PE, blueberry powder or tart cherry powder.

In some embodiments the anti-inflammatory composition contains aglutathione precursor, a vitamin anti-oxidant or an alpha lipoic acid.The composition optionally also contains a trace mineral. Theglutathione precursor is taurine or N-acetyl-L-cysteine. A vitaminanti-oxidant includes for example, Vitamin A, Vitamin B, Vitamin C, orVitamin E, or Vitamin D. Fat soluble ingredients, e.g., vitamin A, D, K,carotenoids, alpha-lipoic acid, and choline, are preferably present inan emulsified form. The composition also includes L-carnitine.Preferably, the composition does not contain histidine or a mucin.

The invention also includes a method of reducing inflammation in anocular tissue Inflammation is inhibited by administering to an inflamedtissue an anti-inflammatory composition described above. An inflamedtissue is characterized by redness, pain and swelling of the tissue. Thetissue includes ocular tissue. For example, the ocular tissue is scleratissue, iris tissue, cornea tissue, pupil tissue, lens tissue,conjuctiva tissue, vitreous tissue, choroids tissue, macula tissue orretina tissue. Optionally, the tissue is contacted with an omega-3 fattyacid such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), oralpha linolenic acid (ALA). The fatty acids are in the form of a fishoil or a plant oil (e.g., flaxseed oil). Preferably, the daily dose ofcopper is less than 1.6 mg. For example, the composition contain 0.1 mg,0.25 mg, 0.5 mg, 1.0 mg, 1.5 mg of copper, and the daily dosage does notexceed 2.0 mg. To avoid toxicity, the daily dose of zince is less than40 mg, e.g., the daily dose is 5, 10, 15, 20, 25, 30, 35 mg. In someembodiments, the composition to be administered contains lutein, e.g.,an emulsified lutein.

Also within the invention is a method of alleviating a symptom of anocular inflammatory disease such as dry eye or macular degeneration byadministering to a subject one or more of the anti-inflammatorycompositions described above. The subject is a mammal, such as human, aprimate, mouse, rat, dog, cat, cow, horse, pig. The subject is sufferingfrom or at risk of developing an ocular inflammatory disease. A subjectsuffering from or at risk of developing an inflammatory eye condition isidentified by methods known in the art, e.g., itching, burningirritation, redness, blurred vision, or difficulty reading. Symptoms ofinflammation include pain, redness and swelling of the affected tissue.A subject suffering from or at risk of developing an inflammatory eyecondition or disease such as age-related macular degeneration (AMD) isalso identified using a standard visual accuity test to detect loss ofcentral vision (with retention of peripheral vision) and/or detecting anelevated level of C-reactive protein compared to a normal control levelof C-reactive protein in serum or blood. In an adult humans, a serumlevel of C-reactive protein in the range of 0.08-3.1 mg/L is a normalreference range for healthy persons. In addition to monitoring symptomslisted above, reduced inflammation and an improvement in the severity ofan inflammatory eye syndrome is also identified by detecting a reducedlevel of serum or blood levels of C-reactive protein over timepost-administration of the compositions described herein.

The composition is administered systemically, e.g., orally.Alternatively, the composition is administered locally. For example, thecomposition is administered by directly contacting an inflamed oculartissue with the composition. The compositions are administered prior toafter development of ocular inflammation as a prophylaxis; or afterdevelopment of ocular inflammation as a therapeutic.

Optionally, the subject is co-administered a composition containing aomega-3 fatty acid and/or omega-6 fatty acid. Preferably, thecomposition contains both omega-3 and omega-6 fatty acids in amountsthat produce a synergistic anti-inflammatory effect.

Also within the invention is a composition containing a carotenoid and apolyphenol, each of which are present in amounts to produce asynergistic anti-inflammatory effect. By synergistic is meant that thecombination of ingredients in a mixture leads to a total effect that isgreater than the sum of the effect produced by the two (or more)ingredients individually. When administered to a subject, theanti-inflammatory composition is associated with reduced adverse sideeffects such as decreased cell-mediated immunity compared side effectsassociated with conventional anti-inflammatory drugs. Theanti-inflammatory composition contains a lipid-soluble antioxidantcarotenoid. In some embodiments, the composition does not contain abeta-carotene compound. The composition may also contain a water-solubleantioxidant (vitamin C or ascorbic acid) and/or a ginkgolide.

Accordingly, the invention provides a composition containing a lipidsoluble antioxidant and a water-soluble antioxidant. The lipid solubleantioxidant is a carotenoid compound. The carotenoid compound isastaxanthin or an ester thereof or a vitamin such as ascorbic acid.Alternatively, the water soluble antioxidant is a ginkgolide such as aterpene trilactone selected from the group consisting of Gingkolide A,Gingkolide B, Gingkolide C, Gingkolide J, Gingkolide M, and bilobalide.The gingkolide composition preferably exhibits one or more of thefollowing activities: (i) platelet activating factor receptor (PAFR)antagonist activity; (ii) PLA2-inhibitory capability; (iii)COX-2-inhibitory capability; and (iv) the capability to inhibit cAMPphosphodiesterase. Preferably, the composition exhibits all of theaforementioned activities. For example, the ginkgolide compositioncontains Egb 761. The composition optionally also contains an histaminerelease inhibitor such as a cetirizine compound and/or an azelastinecompound. In preferred embodiments, the composition contains a mixtureof an asaxanthin compound, a ginkgolide compound, and an ascorbic acidcompound.

The invention also includes a method of suppressing inflammation in amammal. The method is carried out by co-administering of astaxanthin ora derivative thereof, vitamin C, and one or more classes of gingkolidein such amounts so as to provide an additive or synergisticanti-inflammatory effect. Preferably, the gingkolide is administered ata dose that preferentially inhibits expression of an inflammatorycytokine much as IL-8, IL-1α, IL-1β, TNF-α or IL-6.

For example, the invention provides a method of inhibiting activation ofan immune cell by contacting the immune cell (e.g., a T cell or a mastcell) with the composition(s) described above. Also within the inventionis a method of alleviating a symptom of an inflammatory disease byadministering to a mammal suffering from or at risk of developing thedisease one or more of the anti-inflammatory composition describedabove. In one example, the composition is administered systemically.Alternatively, the composition is administered locally. For example, thecomposition is administered by directly contacting an inflammed tissuewith the composition. The tissue to be directly contacted is dermaltissue in the case of skin inflammatory diseases such as psoriasis. Forasthma, the tissue is pulmonary tissue, e.g., bronchoalveolar tissue. Inthe former case, the compositions are administered topically, e.g., bycontacting skin with a cream, lotion, or ointment. In the latter case,pulmonary tissue is contacted by inhaling a composition, e.g., a liquidor powder aspirate containing the mixture of anti-inflammatorycompounds.

Antioxidants such as carotenoids are co-administered with other agentsto reduce inflammation. For example, astaxanthin (or esters thereof),vitamin C, and the gingkolide(s) are administered simultaneously orconsecutively. For example, the gingkolide(s) is first administeredfollowed by astaxanthin, followed by vitamin C. Alternatively,astaxanthin is administered first and then the gingkolide(s) and thenvitamin C. In another regimen, vitamin C is administered first, followedby astaxanthin, followed by the ginkgolide(s); or vitamin C isadministered following administration of either astaxanthin or theginkgolide, followed by administration of the third component. Thecombination of compounds is administered in the presence or absence of atraditional anti-inflammatory agent such as a corticosteroid ornon-steroidal anti-inflammatory agent.

Such a co-administration regimen is useful to inhibit inflammation in amammal. For example, each of the aforementioned three classes ofcompounds is administered prior to after development of inflammation asa prophylaxis; or after development of inflammation as a therapeutic.

The antioxidant and gingkolide compounds described are also useful incombination with nonsteroidal anti-inflammatory drugs (NSAIDs) to reducethe dose of NSAID required to achieve a desired clinical effect such asreduction of symptoms associated with Alzheimer's Disease. Combined withhistamine release blockers such as cetirizine, the antioxidant andgingkolide compounds augment the clinical effect (e.g., reduction ofallergy symptoms such as itching) of the histamine release blocker,thereby permitting administration of a lower dose of the histaminerelease blocker. Coadministration of an antioxidant and/or a gingkolidecompound reduces adverse side effects associated with many knownanti-inflammatory and anti-allergy medications.

Individual ingredients or compounds in the composition are whole foods,e.g., ground cinnamon bark, or purified/isolated compounds from anatural or genetically-engineered source. By purified or isolated ismeant that the desired individual ingredient or compound (prior to itsformulation into the claimed combination product) is 85% of thecomposition by weight (w/w). Preferably, the desired ingredient orcompound is at least 90, 95, 98, 99, or 100% of the composition byweight (w/w) prior to being formulated into the combination product.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the effect of astaxanthin (ASX) on immuneactivation of human PBMC. Cells cultured 24 h at 37° C., 5% CO₂ in RPMI1640, 10% FCS with 50 mg/ml PHA and ASX were evaluated by 3-color flowcytometry for immune activation as % CD3+ cells induced to expressmembrane-bound CD25 (IL-2 receptor). Stimulation indices (SI) weredetermined as the ratio of % CD3+CD25+ cells in fully-stimulatedcultures treated with PHA alone, to those cultured with PHA plus ASX.Results are representative of independent assays conducted on cells of6-8 asthmatic donors participating in this study. Significance incomparison with fully-stimulated cultures: (*:p<0.05)

FIG. 2 is a bar graph showing the effect of ginkgolide B (GB) on immuneactivation of human PBMC. Cells cultured 24 h at 37° C., 5% CO₂ in RPMI1640, 10% FCS with 50 mg/ml PHA and GB were evaluated by 3-color flowcytometry for immune activation as % CD3+ cells induced to expressmembrane-bound CD25 (IL-2 receptor). Stimulation indices (SI) weredetermined as the ratio of % CD3+CD25+ cells in fully-stimulatedcultures treated with PHA alone, to those cultured with PHA plus GB.Results are representative of independent assays conducted on cells of6-7 asthmatic donors participating in this study. Significance incomparison with fully-stimulated cultures: (*:p<0.05)

FIG. 3 is a bar graph showing the effect of astaxanthin (ASX) plusginkgolide B (GB) on immune activation of human PBMC. Cells cultured 24h with 50 mg/ml PHA and selected combinations of ASX+GB were evaluatedby 3-color flow cytometry for immune activation as % CD3+ cells inducedto express CD25 (IL-2 receptor). Stimulation indices (SI) are determinedas the ratio of % CD3+CD25+ cells in fully-stimulated cultures treatedwith PHA alone, to those cultured with PHA plus selected combinations ofASX+GB. Results are representative of independent assays conducted oncells of 4-7 healthy adult donors participating in this study.Significance in comparison with fully-stimulated cultures: (*:p<0.05)

FIGS. 4A-4B are bar graphs showing the effect of cetirizine (Zyrtec/CTZ)versus azalestene (AZE) on immune activation of human PBMC. Cellscultured 24 h at 37° C., 5% CO₂ in RPMI 1640, 10% FCS with 50 mg/ml PHAand either CTZ (4A) or AZE (FIG. 4B) are evaluate by 3-color flowcytometry for immune activation as % CD3+ cells induced to expressmembrane-bound CD25 (IL-2 receptor). Results are representative ofindependent assays conducted on cells of 7-12 asthmatic donors.Significance (P) in comparison with fully-stimulated culture: (*:p<0.05)

DETAILED DESCRIPTION

The compositions described herein are useful to prevent inflammation,and improve the clinical prognosis for patients suffering frominflammatory disease. The combined action of a lipid-soluble carotenoid(principally astaxanthin) with vitamin C and one or more components of aGinkgo biloba extract mediates prevention or suppression ofdisease-associated inflammation.

Astaxanthin

Astaxanthin (3,3′-dihydroxy-4,4′-diketo-B-carotene) is a carotenoidproduced by several natural sources, including: the marine algaeHaematococcus pluvialis; and the pink yeast Xanthophyllomycesdendrorhous. It is obtained directly from either aforementionedorganism; or alternatively by extraction from by-products of crustaceasuch as the Antarctic krill Euphausia superba. Its molecular structureis similar to that of carotenoid beta-carotene, however smalldifferences in structure confer large differences in the chemical andbiological properties of the two molecules. In particular, astaxanthinis superior to beta-carotene in its capacity to scavenge free radicals.It exhibits strong antioxidant properties and confers protection againstlipid peroxidation and oxidative damage of LDL-cholesterol, cellmembranes, cells, and tissues. Beneficial effects mediated byastaxanthin in mammals are known to include: increased boar semen volumeand piglet litter size and survival rate when fed to pigs; augmentationof anti-stress agents administered to farm animals and household pets;improved immunity; and suppression of tumor growth.

Additionally, esterified astaxanthin from Haematococcus pluvialis algalmeal is therapeutic for muscular dysfunction such as exertionalrhabdomyolysis (also known as exertional myopathy, tying-up syndrome,azoturia, or Monday morning sickness) in horses; and forgastrointestinal tract inflammation due to infections by Helicobactersp. bacteria.

Gingkolides

Ginkgo Biloba. is a plant, the leaves, roots, and fruit of which havebeen used for medicinal purposes for centuries. Extracts of variousparts of the plant are commercially available. A gingkolide, or Ginkgobiloba extract contains one or more biologically active components suchas an antioxidant component and an PAFR antagonist component. Forexample, an extract is made from ginkgo leaves and used at aconcentration that contains about 24-25% ginkgo-flavone-glycosides. Theextract may also contain terpenoids such as Egb761 or LI-1370. Forexample, the preparation contains 24% ginkgo-flavone glycosides and 6%terpenoids. The ginkgo-flavone glycosides are sometimes referred to asheterosides. EGb761 is a commercially available leaf extract of Ginkgobiloba, containing: GA, GB, GC, GJ, GM and bilobalide.

Naturally-occurring Ginkgo biloba contains: (A) biflavones such asamentoflavone, bilobetin, sequoiaflavone, ginkgetin, isoginkgetin,Sciadopitysin; (B) flavonol glycosides; (C) terpene trilactones, such asGingkolide A, Gingkolide B, Gingkolide C, Gingkolide J, Gingkolide M andbilobalide; (D) rutin; (E) quercetin; and (F) a 30 kDa Ginkgo bilobaglycoprotein, which reacts with antiserum against beta 1→2xylose-containing N-glycans. Each component or combinations thereof areisolated from crude extracts of the plant using methods known in theart.

Alphabetically-labeled series of ginkgolide derivatives are furthercharacterized as follows. Ginkgolide A (GA) is a leaf extract containingterpene trilactone. This gingkolide is a PAFR antagonist, but has noapparent antioxidant properties. It is also known as BN52020, CAS15291-75-5. Ginkgolide B (GB) is a leaf extract containing terpenetrilactone. It is a PAFR antagonist, with antioxidant properties and maybe referred to as BN52021 or CAS 15291-77-7. GC, ginkgolide C: a terpenetrilactone, leaf extract. Ginkgolide J (GJ) is a leaf extract containingterpene trilactone with PAFR antagonist activity and antioxidantproperties. Ginkgolide M (GM) is a root extract containing terpenetrilactone. This gingkolide has PAFR antagonist activity and antioxidantproperties. Bilobalide (a sesquiterpene trilactone) is primarily anantioxidant. Ginkgo biloba extract (EGb 761) is a clinically safe,nontoxic, and easily produced product with a wide range of applications.

Other extracts and preparation of gingkolides are known in the art,e.g., as described in Chen et al., 1998, Bioorganic & MedicinalChemistry Letters 8:1291 -6.

The gingkolide compositions to be administered are in a form, whichmaximizes ginkgolide bioavailability. For example, the composition is avariation of EGb 761 containing 27% ginkgo-flavonol glycosides, 7%terpene lactones. This composition extends bioavailability ofpharmacologically active ginkgolide components (Li et al, 1997, PlantaMedica. 63:563-5).

Among the compositions to be administered is BN 50730, an analog to theterpene trilactone BN52021 (GB). BN 50730 is a synthetic hetrazepinederivative of BN 52021. It shows a several ten-folds more potent PAFantagonistic activity in vitro than BN52021.

Anti-Inflammatory Drug Combinations

The dose-response curve of astaxanthin in suppression of in vitroexpression of an inflammation-associated cytokine was found to befavorably altered in the presence of a ginkgolide. Inflammatory damageis suppressed by astaxanthin or its derivatives and further reduced byco-administration of a ginkgolide.

The combination drug therapy regimen described herein is based on thepharmacological action of astaxanthin, ginkgolides and vitamin C. Byacting as a powerful scavenger of free radicals, astaxanthin inhibitstissue damage mediated by these chemical species. However, sinceastaxanthin and its derivatives are primarily lipid-soluble, the adductoften remains membrane associated. Effective clearance of freeradical-astaxanthin reaction products is mediated by co-administrationof a water-soluble scavenger of free radicals. For example, thewater-soluble free radical scavenger is vitamin C. Gingkolidecompositions include extracts of ginkgo such as EGb761. The gingkolidealone or in combination with vitamin C; or astaxanthin alone, or incombination with vitamin C; or astaxanthin plus a ginkgolide; orastaxanthin plus a ginkgolide plus vitamin C are used for suppression ofdisease-associated inflammation.

For example, the dose of astaxanthin plus ginkgolide and vitamin Crequired to achieve clinically significant suppression of inflammationis at least 5%, preferably at least 10%, preferably at least 25%,preferably at least 30%, more preferably at least 40%, and mostpreferably at least 50% less than that required for the same level ofsuppression of inflammation in the absence of a gingkolide and vitaminC. Suppression of inflammation is measured using methods known in theart, e.g., by detecting reduced expression of pro-inflammatory cytokinesboth in vitro (cell culture approach) and in vivo (immunohistochemicalapproach), given stimulus of experimental model in a manner known in theart to induce expression of these cytokines.

Toxicity

An astaxanthin/ginkgolide/vitamin C combination drug offers a method forachieving suppression of disease-associated inflammation in a mannersuperior to currently available drugs. Moreover, since each componentexhibits low-to-negligible toxicity levels, and therefore, is applicableto a broad patient population.

Treatment and Alleviation of Symptoms of Inflammatory Disease

Clinical effects of formulations based on co-administration ofastaxanthin plus ginkgolides and/or vitamin C include application toinflammation associated with autoimmune conditions (such as type Idiabetes), asthma, psoriasis and cardiac disorders. These combinationswill also aid in post-organ transplant drug therapy. Suppression ofgraft rejection-associated inflammation by these drugs is sufficient tomaintain transplanted tissue in a healthy, functional state with littleor no side effects.

Advantages of the invention include improved outcomes to transplantsurgery (both in terms of survival as well as drug-related morbidity),decreased need for secondary hospitalization, and reduced expenditure ofhealth care costs for transplant recipents.

The coadministration strategy also decreases the incidence ofischemia/reperfusion-related damage to organs occurring postoperatively,or as a result of ischemic disease as a result of the capacity of theseformulations to inhibit basic inflammatory processes.

Platelet Activating Factor (PAF)/Calcium-Dependent Protection andMechanisms of Inflammation

Cellular signaling pathways resulting in inflammatory responses aredependent largely upon receptor-mediated release of calcium stores (suchas within the endoplasmic or sarcoplasmic reticulum), followed byexpression of inflammatory mediators. This calcium availability may bereduced by treatment of a subject one or more subcomponents of Ginkgobiloba (e.g., EGb761). The gingkolide acts as an antagonist to thereceptor for PAF, a potent bioactive phospholipid. The PAFR, whenengaged by PAF, activates a signalling pathway causing a rise inintracellular calcium. Gingkolide compounds inhibit PAF-mediatedincrease in cytoplasmic calcium, in turn suppressing release ofeicosonoids, pro-inflammatory cytokines, free radical species and othermajor mediators of inflammation.

Prevention of PAF/COX-2-Mediated Effects

PAF stimulates transcription of COX-2 (inducible prostaglandinsynthase), which contributes to inflammatory damage. Ischemia of anytissue promotes PAF overproduction. PAF activity is blocked withginkgolides exhibiting PAF receptor antagonist properties.

Amplification of Pharmacological Effect by Increasing GinkgolideBioavailability

EGb 761 is a standardized extract of dried leaves of Ginkgo bilobacontaining 24% ginkgo-flavonol glycosides, 6% terpene lactones (24/6)such as ginkgolides A, B, C, J and bilobalide. The PAFR antagonistic andantioxidant effects of EGb761 confers clinical benefit, alone or whencombined with astaxanthin and/or vitamin C. For example, animmunosuppressive compound contains a calcineurin inhibitor with extractof Ginkgo biloba with a ratio of 27% ginkgo-flavonol glycosides, 7%terpene lactones (27/7), enriched in ginkgolide B. Preparation of thegingkolide portion of the composition is known in the art, e.g., themethod of Li, et al., 1997, Planta Medica. 63(6):563-5.

Therapeutic Administration

The results indicate that the combination of astaxanthin and agingkolide is useful to inhibit inflammatory damage occurring as aresult of a diverse range of diseases. The compositions are formulatedinto therapeutic compositions such as liquid solutions, emulsions, orsuspensions, tablets, pills, powders, suppositories, polymericmicrocapsules or microvesicles, liposomes, and injectable, eye drops orinfusible solutions. The preferred form depends upon the mode ofadministration and the particular indication targeted. The compositionsalso include pharmaceutically acceptable vehicles or carriers. Suitablevehicles are, for example, water, saline, dextrose, glycerol, ethanol,or the like, and combinations thereof. Actual methods of preparing suchcompositions are known to those skilled in the art (e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18thedition, 1990). The compositions are administered in one or more units,e.g., pills, tablets, or capsules. For example, one unit contains thecomposition described in Table A or B, and a second unit containsanother composition or combination, e.g., one containing greater than1/16 tsp. of ground cinnamon.

The compositions are administered using conventional modes of deliveryincluding intravenous, intraperitoneal, oral or subcutaneousadministration. In addition to systemic administration, the compositionsare locally administered, e.g., to the site of inflammation.

The dosages of astaxanthin and of gingkolide and vitamin C may varydepending on the severity and course of the disease, the patient'shealth and response to treatment, and the judgment of the treatingphysician.

Astaxanthin, vitamin C and the gingkolide are administeredsimultaneously or sequentially. Astaxanthin dosages range from 0.1-4.0g/kg body weight per day; gingkolide compositions are administered indoses of 0.1 mg/kg/day to 1000 mg/kg/day. (e.g., 10 mg/kg/day-60mg/kg/day); and dosage of vitamin C will include regimens of 1.0-400.0mg/kg/day. Routes of administration are comparable to those used forimmunophilin-binding compounds such as calcineurin inhibitors. Forprevention or treatment of inflammatory eye conditions, astaxanthin isadministered at a dose of less than 5 mg/day, e.g., astaxanthin isadministered in a dose of 0.254.9 mg/day, and zeaxanthin is administeredin a dose range of 0.1-2.9 mg/day, e.g., at dose of about 1.0 mg/day.

For prevention and/or treatment of an inflammatory eye condition,omega-3 fatty acids are administered in a dose range of 100-1500 mg/day,e.g., about 1000 mg/day. Similarly, omega-6 fatty acids are administeredin a dose range of 100-1500 mg/day e.g., about 1000 mg/day. Preferably,the composition contains both omega-3 and omega-6 fatty acids in amountsthat produce a synergistic anti-inflammatory effect in ocular tissues,e.g., 180 mg/day of EPA and 120 mg/day of DHA. A plant-based omega-3fatty acid such as ALA is administered in a dose range of 200-3000,e.g., 500 mg/day.

The compositions also optionally contain acetyl L-carnitine. L-carnitineis administered in a daily dose of 100-1500 mg/day, e.g., 1000 mg/day.For example, L-carnitine is administered at a dose of 1000 mg/day.

The compositions are administered as prophylaxis to prevent onset of aninflammatory condition, or before or after development of disease.Subjects to be treated is include those who have been diagnosed ashaving a condition characterized by aberrant immune activation (e.g.,pathological T cell activation or pathological inflammation), those whoare at risk of developing such a condition, and those who have apersonal or family history of such a condition. Such aberrantinflammatory events include an asthma attack. Methods for diagnosis areknown in the art. For example, the anti-inflammatory compositions areuseful to treat or prevent autoimmune disease and/or inflammatoryconditions such as asthma, arthritis (e.g., rheumatoid arthritis,arthritis chronic progrediente and arthritis deformans) and rheumaticdiseases. Specific auto-immune diseases for which the compositions ofthe invention may be employed include, autoimmune hematologicaldisorders (including e.g. hemolytic anaemia, aplastic anaemia, pure redcell anaemia and idiopathic thrombocytopenia), systemic lupuserythematosus, polychondritis, sclerodoma, Wegener granulamatosis,dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis,Steven-Johnson syndrome, idiopathic sprue, autoimmune inflammatory boweldisease (including e.g. ulcerative colitis and Crohn's disease)endocrine ophthalmopathy, Graves disease, sarcoidosis, multiplesclerosis, primary billiary cirrhosis, juvenile diabetes (diabetesmellitus type I), uveitis (anterior and posterior), keratoconjunctivitissicca and vernal keratoconjunctivitis, interstitial lung fibrosis,psoriatic arthritis, glomerulonephritis (with and without nephroticsyndrome, e.g. including idiopathic nephrotic syndrome or minimal changenephropathy) and juvenile dermatomyositis.

Individuals to be treated include any member of the class Mammalia,including, humans and non-human primates, such as chimpanzees and otherapes and monkey species; farm animals such as cattle, sheep, pigs, goatsand horses; domestic mammals such as dogs and cats; and laboratoryanimals including rodents such as mice, rats and guinea pigs.Preferably, the mammal is not a rodent such as a rat. The compositionsand methods are suitable for treatment of adult, newborn and fetalmammals. Treatment encompasses the prevention of and adverse clinicalconditions and the reduction or elimination of symptoms of a disease oradverse clinical condition. An anti-inflammatory composition refers toany composition that suppresses or prevents an undesired inflammatoryresponse, e.g., prevents pain, tissue damage and disfigurement.

The combination drug therapy described herein utilizes astaxanthinand/or its derivatives; and a gingkolide composition, which containsPAFR antagonist activity and antioxidant activity. Preferably, thegingkolide compositions contains at least two antioxidant components ofGingko biloba, e.g., GB, GC, GJ, or GM, rather than one component suchas GM alone. For example, the gingkolide composition is Egb761 containsseveral antioxidant components of Gingko biloba in addition to acomponent with PAFR antagonist activity. EGb761 contains a full range ofantioxidants and PAFR antagonists produced by leaves of the plant.

Anti-Inflammatory Combinations Useful for Protecting Ocular Tissue fromInflammatory or Oxidative Damage

The anti-inflammatory composition contains a carotenoid (e.g.,astaxanthin or zeaxanthin) and a polyphenol. Zeaxanthin and lutein areisolated or purified from plant tissues, e.g., genetically-engineeredplant or algae sources. For example, the zeaxanthin used in thecompositions described herein is purified or isolated from orange bellpeppers. Optionally, the composition contains a glutathione precursor, avitamin anti-oxidant an alpha lipoic acid or a trace mineral(hydromins). Additionally, the composition contains an inhibitor ofstress induced mediated tissue damage, an inhibitor of pro-inflammatoryprostaglandin (e.g., gamma linoleic acid or omega-3 fatty acid) or aninhibitor of NFkB (e.g., selenium, N-acetyl-L-cysteine, quercetin orbioflavonoids) or one or more essential vitamins or minerals. Anessential vitamin or mineral is a vitamin or mineral that the bodycannot synthesize itself. These combination are herein referred to as“Ocular protective compositions or OPC combination”

A polyphenol is curcuma longo root powder, green tea, grape seedextract, or cinnamon powder, citrus bioflavonoid. Alternatively, thepolyphenol is a cox-2 inhibitor compound. Exemplary cox-2 inhibitorcompounds include quercetin, bilberry extract, hops PE, blueberry powderor tart cherry powder.

For example, the polyphenol in the composition is a water-solublepolyphenol polymer, e.g., type A polyphenol, from cinnamon. The cinnamoncomponent is derived from any member of the Cinnamomum genus, e.g., C.verum or C. cassia. Preferably, the composition contains Korintje orSaigon cinnamon. The composition contains whole ground cinnamon bark orleaves purified volatile oils, e.g., cinnamaldehye and eugenol, and/orpurified water-soluble flavonoids or polyphenols. Cinnamon-derivedpolyphenols function as antioxidants and include polymers composed ofmonomeric units with a molecular mass of 288 as well as a trimer with amolecular mass of 864, and a tetramer with a mass of 1152 which areisolated from one another using known methods. These polyphenolicpolymers are type-A doubly linked procyanidin oligomers of the catechinsand/or epicatechins. Cinnamon-derived anti-inflammatory compositions arepreferably in the form of ground cinnamon powder. The powder containswater-soluble polyphenolic flavonoids as well as volatile oils (e.g., inamounts of at least 1, 2, 3, and up to 5%). Cinnamon is administered ata dose of 1/16-1 teaspoon (tsp.) per day. Preferably, the dose is ⅛-¼tsp. per day. Optionally, the cinnamon powder is administered in aseparate unit dose, e.g., a pill or capsule, containing greater than1/16 tsp. of cinnamon powder.

A glutathione precursor is for example taurine or N-acetyl-L-cysteine.Taurine is an amino acid-like compound and is a component of bile acids.Taurine are used to help absorb fats and fat-soluble vitamins.N-acetyl-L-cysteine is a free-radical scavenger.

A vitamin anti-oxidant includes Vitamin A (e.g., beta carotene), VitaminB, Vitamin C (e.g., ascorbic acid) Vitamin D, or Vitamin E. Vitamin B isa group of eight vitamins, which include thiamine (B1), riboflavin (B2),niacin (B3), pyridoxine (B6), folic acid (B9), cyanocobalamin (B12),pantothenic acid and biotin. Vitamin E is a mixture of tocopherols andtocotrienols. Tocopherols include alpha-tocopherol, beta-tocopherol,gamma-tocopherol and delta-tocopherol. Tocotrienols includealpha-tocotrienol beta-tocotrienol gamma-tocotrienol delta-tocotrienol.

For example an anti-inflammatory composition contains a vitamin Apalmitate compound, an ascorbic acid compound, a mixed tocopherolcompound, an alpha lipoic compound, a polyphenol compound, ananthocyanidin compound, a blueberry compound, a ginkgo biloba compound,a hops PE compound, a quercetin compound, a tocotrienol complexcompound, a N-acetyl-L-cysteine compound a curcuma longa root compound,zeaxanthin compound, an astaxanthin compound, and a tart cherrycompound. Optionally, the composition contains one or more of thefollowing compounds, a beta carotene compound-alpha tocopheryl succinatecompound. A pyridxine HCl compound, a folic acid compound, a zinccitrate compound, a grape seed extract compound, a citrus bioflavonoidcompound, a taurine compound, zeaxanthin compound, a mixed carotenoidcompound, and a hydromins compound a grape seed extract compound, acitrus bioflavonoid compound, a taurine compound, zeaxanthin compound, amixed carotenoid compound, or a hydromins compound.

Exemplary anti-inflammatory compositions include the formulations ofTable A and B shown below. Table C shows a daily dose range for eachingredient. Dosages are for administration to a human adult. TABLE ALABEL CLAIM INGREDIENT NAME DAILY DOSAGE 62.5 IU Vitamin A (as Vitamin APalmitate) 250.00 IU 1250 IU Vitamin A (as Beta Carotene) 5000.00 IU 75MG Vitamin C (as Ascorbic Acid) 300.00 MG 60 IU Vitamin D (asCholecalciferol) 200.00 IU 25 IU Vitamin E (as d-alpha TocopherylSuccinate) 100.00 IU 12.5 IU Vitamin E (as Mixed Tocopherols) 50.00 IU25 MG Vitamin E (as gamma Tocopherol) 100 MG 0.015 MG Vitamin K 0.06 MG0.3 MG Thiamin (as Thiamine Mononitrate) 1.20 MG 0.326 MG Riboflavin1.30 MG 4 MG Niacin (as Niacinamide) 16.00 MG 0.75 MG Vitamin B6 (asPyridoxine HCl) 3.00 MG 0.1 MG Folate (as Folic Acid) 0.40 MG 0.006 MGVitamin B12 (as Cyanocobalamin) 0.0024 MG 1.25 MG Pantothenic Acid (asCalcium Pantothenate) 5.00 MG 0.0075 MG Biotin 0.03 MG 68.75 MG Choline(as Choline Bitartrate) 275.00 MG 0.025 MG Chromium (as Chromiumnicotinate) 0.10 MG 0.5 MG Copper (as Copper Citrate) 2.00 MG 0.0375 MGIodine (as Potassium Iodine) 0.15 MG 25 MG Magnesium (as MagnesiumCitrate) 100.00 MG 0.575 MG Manganese (as Manganese Citrate) 2.30 MG0.025 MG Selenium (as Selenomathionine) 0.10 MG 3.75 MG Zinc (as ZincCitrate) 15.00 MG 7.5 MG Alpha Lipoic Acid 30.00 MG 50 MG Green Tea (40%Polyphenols) 200.00 MG 1 MG Bilberry Ext (25% Anthocyanidins) 4.00 MG 25MG Blueberry Powder 100.00 MG 62.5 MG Ginkgo biloba SE 24/6 250.00 MG2.5 MG Hops PE 10.00 MG 12.5 MG Quercetin 50.00 MG 12.5 MG TocotrienolComplex 50.00 MG 5 MG Grape Seed Extract 20.00 MG 100 MG CitrusBioflavonoids 400.00 MG 75 MG Taurine 300.00 MG 50 MGN-Acetyl-L-Cysteine 200.00 MG 12.5 MG Curcuma longa Root Powder 50.00 MG0.25 MG Zeaxanthin 1.00 MG 0.25 MG Astaxanthin 1.00 MG 12.5 MG MixedCarotenoids 50.00 MG 12.5 MG Trace Minerals (Hydromins) 50.00 MG 12.5 MGTart Cherry Powder 50.00 MG Excipients 56 MG Croscarmellose Sodium224.00 MG 14 MG Magnesium Stearate 56.00 MG 24 MG Silicon Dioxide 96.00MG 28 MG Stearic Acid 112.00 MG 210 MG Microcrystalline Cellulose 840.00MG 40.16 MG Color Coating (3% target weight)

TABLE B LABEL CLAIM INGREDIENT NAME DAILY DOSAGE 125 IU Vitamin A (asVitamin A Palmitate) 250.00 IU 2500 IU Vitamin A (as Beta Carotene)5000.00 IU 150 MG Vitamin C (as Ascorbic Acid) 300.00 MG 50 IU Vitamin E(as d-alpha Tocopheryl Succinate) 100.00 IU 25 IU Vitamin E (as MixedTocopherols) 50.00 IU 50 MG Vitamin E (as gamma Tocopherol) 100 MG 1.6MG B6 (as Pyndoxine HCl) 3.00 MG 0.2 MG Folate (as Folic Acid) 0.40 MG7.5 MG Zinc (as Zinc Citrate) 15.00 MG 15 MG Alpha Lipoic Acid 30.00 MG100 MG Green Tea (40% polyphenols) 200.00 MG 0.5 MG Astaxanthin 1.00 MG125 MG Ginkgo biloba SE 24/6 250.00 MG 5 MG Hops PE 10.00 MG 25 MGQuercetin 50.00 MG 25 MG Tocotrienol Complex 50.00 MG 2 MG Bilberry Ext(25% Anthocyanidins) 4.00 MG 25 MG Blueberry Powder 50.00 MG 25 MG TartCherry 50.00 MG 25 MG Curcuma longa Root Powder 50.00 MG 100 MGN-Acetyl-L-Cysteine 200.00 MG Excipients 48 MG Croscarmellose Sodium96.00 MG 12 MG Magnesium Stearate 24.00 MG 14 MG Silicon Dioxide 28.00MG 25 MG Stearic Acid 50.00 MG 200 MG Microcrystalline Cellulose 400.00MG 35.34 MG White Coating 3% target weight)

TABLE C Daily Dosage Range DAILY DOSAGE INGREDIENT NAME RANGE Vitamin A(as Vitamin A Palmitate)  100-1000 IU Vitamin A (as Beta Carotene)2000-7500 IU Vitamin C (as Ascorbic Acid) 150-500 MG Vitamin D (asCholecalciferol) 150-400 IU Vitamin E (as d-alpha Tocopheryl Succinate) 75-200 IU Vitamin E (as Mixed Tocopherols)  25-200 IU Vitamin E (asgamma tocopherol)  5-200 IU Vitamin K 0.03-0.08 MG Thiamin (as ThiamineMononitrate)   1-2.2 MG Riboflavin   1-2.3 MG Niacin (as Niacinamide)10-26 MG Vitamin B6 (as Pyridoxine HCl) 2-4 MG Folate (as Folic Acid)0.2-0.5 MG Vitamin B12 (as Cyanocobalamin) 0.002-0.004 MG PantothenicAcid (as Calcium Pantothenate) 2.5-6   MG Biotin 0.02-0.06 MG Choline(as Choline Bitartrate) 200-400 MG Chromium (as Chromium nicotinate)0.07-0.15 MG Copper (as Copper Citrate) 1.0-1.6 MG Iodine (as PotassiumIodine) 0.1-0.2 MG Magnesium (as Magnesium Citrate)  75-200 MG Manganese(as Manganese Citrate) 2-3 MG Selenium (as Selenomathionine) 0.07-20  MG Zinc (as Zinc Citrate) 10-40 MG Alpha Lipoic Acid  20-100 MG GreenTea (40% Polyphenols)  50-500 MG Bilberry Ext (25% Anthocyanidins)  2-20MG Blueberry Powder  20-500 MG Ginkgo biloba SE 24/6  50-300 MG Hops PE 1-100 MG Quercetin  10-200 MG Tocotrienol Complex  10-200 MG Grape SeedExtract  5-100 MG Citrus Bioflavonoids 100-600 MG Taurine  50-500 MGN-Acetyl-L-Cysteine  50-400 MG Curcuma longa Root Powder  10-200 MGZeaxanthin 0.1-2.9 MG Astaxanthin 0.01-4.9  MG Mixed Carotenoids  10-100MG Trace Minerals (Hydromins)  10-100 MG Tart Cherry Powder  10-200 MGReduction of Ocular Inflammation and Treatment of Ocular InflammatoryDisease

Inflammation is inhibited by administering to tissue an OPC combinationdescribed above. Optionally, the tissue is contacted with aomega-3-fatty acid such as eicosapentaenoic acid or docosahexaenoicacid.

Tissues to be treated include ocular tissue such as sclera tissue, iristissue, cornea tissue, pupil tissue, lens tissue, conjuctiva tissue,vitreous tissue, choroids tissue, macula tissue or retina tissue.Inhibition of inflammation is characterized by a reduction of redness,pain and swelling of the treated tissue compared to a tissue that hasnot been contacted with an OPC combination. OPC combinations areadministered in an amount sufficient to decrease (e.g., inhibit)inflammatory cytokine production. An inflammatory cytokine is a cytokinethat modulates, e.g., induces or reduces an inflammatory response. Aninflammatory response is evaluated by morphologically by observingtissue damage, localized redness, and swelling of the affected area. Aninflammatory cytokine is a proinflammatory cytokine. For example theinflammatory cytokine is, TNF alpha, interferon (e.g., alpha, beta orgamma), or interleukin (e.g., IL-1, IL-6, IL-10, IL-12, IL-14, IL-18).Cytokines are detected for example in the serum, plasma or the tissue.Cytokine production is measured by methods know in the art.

Tissues are directly contacted with an OPC combination. For example theOPC combination is applied directly to the eye. Alternatively, the OPCcombination is administered systemically. An inflammatory response isevaluated by morphologically by observing tissue damage, localizedredness, and swelling of the affected area.

The methods are useful to alleviate the symptoms of a variety of ocularinflammatory disorders. The ocular inflammatory disorder is acute orchronic. Ocular inflammatory disorders include dry eye, or maculardegeneration.

The methods described herein lead to a reduction in the severity or thealleviation of one or more symptoms of an ocular inflammatory disordersuch as those described herein. Ocular inflammatory disorders arediagnosed and or monitored, typically by a physician using standardmethodologies. Alleviation of one or more symptoms of the ocularinflammatory disorder indicates that the compound confers a clinicalbenefit. A reduced risk of developing macular degeneration is alsoidentified by detecting a reduction in the level of C-reactive proteinafter administration of the OPC compositions described above. Methodsfor measuring C-reactive protein and age-related levels are known in theart (e.g., Hutchinson et al., 2000, Clin. Chem. 46:934-8). For example,blood or serum levels are measured 24 hours, 2 days, 5 days, 1 week, 2weeks, 1 month, 2 months, 3 months, 6 months, 12 months or more afterthe initiation of an OPC treatment regimen to evaluate clinical status.A decrease in C-reactive protein levels over time indicates an improvedcondition and positive prognosis. The compositions are also useful toreduce homocysteine levels in an individual. A decrease in homocysteinlevels over time or over the course of OPC therapy indicates an improvedcondition and positive prognosis.

Dry eye syndrome is one of the most common problems treated by eyephysicians. Over ten million Americans suffer from dry eyes. It isusually caused by a problem with the quality of the tear film thatlubricates the eyes. Dry eye syndrome has many causes. One of the mostcommon reasons for dryness is simply the normal aging process. As wegrow older, our bodies produce less oil −60% less at age 65 then at age18. This is more pronounced in women, who tend to have drier skin thenmen. The oil deficiency also affects the tear film. Without as much oilto seal the watery layer, the tear film evaporates much faster, leavingdry areas on the cornea. Other factors, such as hot, dry or windyclimates, high altitudes, air-conditioning and cigarette smoke alsocause dry eyes. Contact lens wearers also suffer from dryness becausethe contacts absorb the tear film, causing proteins to form on thesurface of the lens. Certain medications, thyroid conditions, vitamin Adeficiency, and diseases such as Parkinson's and Sjogren's also causedryness. Women frequently experience problems with dry eyes as theyenter menopause because of hormonal changes.

Symptoms of dry eye include itching, burning irritation, redness,blurred vision that improves with blinking, excessive tearing, increaseddiscomfort after periods of reading, watching TV, or working on acomputer.

There are several methods to test for dry eyes. For example, theunderlying cause of the dry eyes will be determined by measuring theproduction evaporation rate and quality of the tear film. Special dropsthat highlight problems that would be otherwise invisible areparticularly helpful to diagnose the presence and extent of the dryness.

Macular degeneration is a degenerative condition of the macula (centralretina) It is the most common cause of vision loss in the United Statesin those 50 or older, and its prevalence increases with age. Maculardegeneration is caused by hardening of the arteries that nourish theretina. This deprives the sensitive retinal tissue of oxygen andnutrients that it needs to function and thrive. As a result, the centralvision deteriorates. Macular degeneration varies widely in severity. Inthe worst cases, it causes a complete loss of central vision, makingreading or driving impossible. For others, it may only cause slightdistortion. Macular degeneration does not cause total blindness since itdoes not affect the peripheral vision.

Macular degeneration is classified as either wet (neovasular) or dry(non-neovasular) About 10% of patients who suffer from maculardegeneration have wet AMD. This type occurs when new vessels form toimprove the blood supply to oxygen-deprived retinal tissue. However, thenew vessels are very delicate and break easily, causing bleeding anddamage to surrounding tissue. Macular degeneration is caused by tovariety of factors. Genetics, age, nutrition, smoking, and sunlightexposure all play a role.

Symptoms of macular degeneration include loss of central vision,difficulty reading or performing tasks that require the ability to seedetail, distorted vision Eye physicians usually diagnose maculardegeneration. Methods of diagnosis, include for example, vision testingamsler grid test, ophthalmoscopy, fundus photography, and fluoresceinangiography.

The OPC combinations are formulated into therapeutic compositions toalleviate one or more symptoms of dry eye or macular degenerationfollowing administration. The compositions are administered usingconventional modes of delivery including intravenous, intraperitoneal,oral or subcutaneous administration. Additionally the compositions arelocally administered, e.g., to the eye.

The dosages of OPC combinations vary depending on the severity andcourse of the ocular inflammatory disorder. Typically, a dosage regimensincludes one or two tablets administered orally twice a day. Preferablythe OPC therapeutic compositions are administered with omega-3 fattyacids, omega-6 fatty acids, or both. Alternatively, the omega-3-fattyacids are administer prior to or after administration of the OPCtherapeutic compositions.

Reactive Oxygen Free Radicals and the Pathogenesis of Dry Eye

Reactive oxygen species (ROS) expressed primarily by leukocytesinfiltrating affected tissue are major mediators of tissue damage ininflammation. Hence antioxidants suppress these effects. A majoretiologic factor in the age-related form of dry eye is endogenouslipophilic and cationic compound N-retinyl-N-retinylidene ethanolamine(A2E) which mediates formation of reactive oxygen and nitrogen freeradical species. ROS are substantially upregulated by T lymphocytesduring activation; moreover blocking this enhancement with antioxidantssuch as glutathione may suppress the activation process. Ginkgo bilobacontains several compounds with known antioxidant capability includingGinkgolide B, C, J and M and Bilobalide. Astaxanthin is one of thestrongest naturally occurring free radical scavengers known, with theadditional benefit of low toxicity combined with a capacity to stabilizenormal immune activity.

Phospholipase A2 (PLA-2) and the Pathogenesis of Dry Eye

The action of PLA-2 on membrane lipids of macrophages and PMNs duringinflammatory processes causes activation of COX-2 and inducible nitricoxide synthetase (iNOS), both of which contribute to tissue destructionand pain in dry eye. The biflavone gingkgolides bilobetin and ginkgetinare potent inhibitors of PLA-2 and contribute to the observed capacityof Ginkgo biloba to ameliorate dry eye symptoms.

Cytokine-Inducible Nitric Oxide Synthetase (iNOS) and the Pathogenesisof Dry Eye

Among the major cytokine-mediated effects is expression of induciblenitric oxide synthase (iNOS/NOS-2) by activated macrophages at highlevels and at lower but still significant levels by several secretory(including lacrimal gland) epithelial cell types under the influence ofTNF-α and IL-1β, causing increased nitric oxide, a process may be asignificant pathophysiological pathway of dry eye syndrome. Thebiflavone gingkgolides bilobetin and ginkgetin are potent inhibitors ofiNOS via their inhibitory effect on phospholipase A-2 (PLA-2) andcontribute to the observed capacity of Ginkgo biloba to ameliorate dryeye symptoms.

Inducible Cyclooxygenase (COX-2) and the Pathogenesis of Dry Eye

Ocular inflammation in dry eye occurs in part due to breach of theblood-ocular barrier and the attraction of macrophages, PMNs and otherleukocytes to affected tissue. This process is mediated substantially byrelease of inflammatory metabolites such as prostaglandins both fromocular tissue and from emigrant leukocytes. A major contributor to thisprocess is the inflammation-induced enzyme cyclooxygenase-2 (COS-2)which has been demonstrated in many ocular tissues including cornealepithelium and endothelium and pigmentary epithelium). Inhibitors ofCOX-2 ameliorate ocular inflammation and pain as well as contributing tomaintenance of a good mydriasis during surgery and control ofpostoperative cystoid macular edema. The biflavone gingkgolidesbilobetin and ginkgetin inhibit COX-2 via their inhibitory effect onphospholipase A-2 (PLA-2) leading to an improvement in one or moresymptoms of dry eye and/or macular degeneration.

Eosinophils and cAMP-Phosphodiesterase and the Pathogenesis of Dry Eye

Eosinophils are a major component of the inflammatory infiltratecharacteristic of dry eye and a major contributor to inflammatory damagein the disorder. Studies in a histamine-induced guinea pig eye model oftissue eosinophilia indicate that oral treatment of the animals withrolipram, an isozyme IV-selective inhibitor of cAMP-specificphosphodiesterase significantly suppressed infiltrate of these cells.The biflavone ginkgolides also exhibit varying capacity to inhibitcAMP-phosphodiesterase, with the degree of enzyme inhibition followingthe order:amentoflavone>bilobetin>sequoiaflavone>ginkgetin=isoginkgetin; butalmost no capacity for inhibition of this enzyme by sciadopitysin.

Omega-3, Omega-6 Fatty Acids

Omega 3 fatty acids (EPA, DHA, fish oil) in the form of dietarysupplements and/or fish consumption (e.g., 4 times/week) offersprotection against acquiring AMD as well as decreasing the progressionof AMD and other dry eye syndromes. DHA is the most prevalent omega 3 inthe human retina (and brain), and is required for proper development ofthe visual system, as well as offering protection against UV damage tothe retina. Cold water fish consumed 4 times/week supply about 4 gramsof EPA/DHA; the equivalent amount of omega fatty acids in a dietarysupplement, e.g., in the combination formulations described herein,reduces the risk of developing and inhibits the progression of AMD andother dry eye syndromes. Plant based omega 3's (alpha linolenicacid-ALA), e.g., in the form of flaxseed oil, are also protective andreduce the risk of developing AMD and reduce the symptoms of dry eyesyndromes. Anti-inflammatory doses are in the range of 0.1-10 mg/day,preferably about 1-2 grams/day for treating or preventing AMD and dryeye syndromes.

EXAMPLE 1 Administraton of Astaxanthin Leads to Suppression ofInflammation

In vitro studies indicate that astaxanthin suppresses expression ofinflammation-associated T cell surface antigens in PMA/I-treated humanPBMC. Cells isolated from whole blood of healthy volunteers werecultured in 96-well plates (2×10⁶/ml) for 24 hours in RPMI 1640, withphorbol 12-myristate 13-acetate (PMA: 25 ng/ml) in conjunction withionomycin, or media; or with PMA/I plus astaxanthin (10⁻⁵ M).

Following incubation, cultured cells were immunofluorescently labeledwith monoclonal antibodies specific for CD3 (T lymphocytes) and the cellsurface antigens CD25 and CD54 which are known to be upregulated in vivoduring both immune activation and during inflammatory processes.Analysis of blood for representation by selected lymphocytesubpopulations was conducted by two-color flow cytometry. Astaxanthinalone significantly inhibited each of the activated T cell phenotypes(Table 1A and 1B). TABLE 1A (Subject A) Stimulation % CD3+CD54+ %CD3+CD54+ Conditions cells cells Unstimulated 1.7 2.7 PMA/I 56.3 66.3Astx 10-7 M 8.4 10.6 Astx 10-6 M 4.2 3.3

TABLE 1B (Subject B) Stimulation % CD3+CD54+ % CD3+CD54+ Conditionscells cells Unstimulated 2.2 1.6 PMA/I 49.5 54.6 Astx 10-7 M 16.2 21.9Astx 10-6 M 6.3 10.3

EXAMPLE 2 Expression of TNF-α by Human PBMC in Vitro is Suppressed byAstaxanthin but not BN52021 and is Suppressed Maximally with AstaxanthinPlus BN52021

PBMC (2×10⁶/ml) from 2 donors were stimulated with 50 μg/ml, PHA; orwith astaxanthin (10⁻⁶ M); or with the ginkgolide BN52021 (GB) (10⁻⁴ M);or with a combination of astaxanthin (10⁻⁶ M) plus GB (10⁻⁴ M); or withmedia. Cells were cultured 24 hours at 37° C., 5% CO₂ and analyzed byELISA for supernatant concentration of TNF-α. Each data point is themean of triplicate samples. Results show that TNF-α expression by PBMCwas significantly increased relative to unstimulated control cultures asa result of PHA stimulation; and was suppressed by astaxanthin, but notGB treatment. As shown in Table 2, the combined treatment with bothastaxanthin and GB suppressed expression of this pro-inflammatorycytokine below that of astaxanthin alone. TABLE 2 Stimulation TNF-alphaStandard Conditions pg/ml Deviation Unstimulated 164.52 21.73 PHA, 50μg/ml 1676.75 103.57 Astx 10−⁶ M 781.34 186.17 BN52021 10−⁴ M 1689.21218.15 Astx 10−⁶ M + BN52021 225.76 52.97 10−⁴ M

EXAMPLE 3 Compositions Containing a Biflavonoid Ginkgolide, Astaxanthin,Vitamin C, and/or an NSAID Suppress Onset of Alzheimer's Disease

Elevation of intracellular cAMP increases the recovery of APP alpha, thephysiological alpha-secretase-derived product of beta APP processing,and concomittantly lowers the production of the pathogenicbeta/gamma-secretase-derived A beta fragment (A42). The pathogenesis ofAlzheimer's disease correlates with altered production, aggregation anddeposition in neuronal tissue of the A peptide, a proteolytic fragmentof 40-42 residues derived from APP. The longer isoform, A42, isselectively increased in the disease and its presence promote productionof beta-amyloid deposits. Beta amyloid in turn induces free radicalproduction, increased glucose uptake, apoptosis and death of nervecells. Extract of Ginkgo biloba (EGb 761) inhibits, in a dose-dependentmanner, the formation of beta-amyloid-derived diffusible neurotoxicsoluble ligands (ADDLs) involved in the pathogenesis of Alzheimer'sdisease. The mechanism for this protective effect involves elevation ofneuronal cAMP which occurs as a result of the cAMPphosphodiesterase-inhibitory properties of the biflavonoid components ofGinkgo biloba.

NSAIDs ibuprofen, indomethacin and sulindac sulphide preferentiallydecrease the highly amyloidogenic A42 peptide (the 42-residue isoform ofthe amyloid-peptide) produced from a variety of cultured cells by asmuch as 80% independently of COX activity. Significant gastrointestinaland renal toxicity associated with long-term COX-1 inhibition limit theclinical utility of current NSAIDS as A42-lowering agents. Because theA42 effect is independent of COX activity, compounds (e.g., thecombinations described herein) with optimized A42 reduction and littleto no inhibition of COX-1 activity are useful for the prevention oralleviation of symptoms associated with Alzheimer's Disease. Such agentsrepresent a new generation of ‘anti-amyloid’ drugs that selectivelytarget production of the highly amyloidogenic A42 species withoutinhibiting either COX activity or the vital physiological functions.

Sustained high dosage, non-steroidal, anti-inflammatory drugs (NSAIDs)inhibit onset of Alzheimers disease, but the dosage required to suppressthe disease is toxic.

Biflavonoid components of ginkgo biloba represent a new generation ofanti-amyloid drugs which, when used in combination with NSAIDs lower theeffective NSAID dosage to subtoxic levels, thereby enabling them to beused to prevent Alzheimer's disease at little or no risk to the generalhealth of the patient.

Astaxanthin and vitamin C contribute to suppression of alzheimersdisease in a manner synergistic with combinations of ginkgobiflavoinoids by suppressing disease associated inflammation, primarilyas free radical scavengers.

The compositions described herein are useful to prevent onset ofAlzheimers disease by inhibiting formation of Beta amyloid plaques as aresult of the combined action of the NSAIDs ibuprofen, indomethacin andsulindac sulphide, (and/or other drugs which act through COX-2inhibition); plus the biflavonoid ginkgolides: amentoflavone, bilobetin,sequoiaflavone, ginkgetin and isoginkgetin. Inflammation associated withAlzheimers is suppressed by combining NSAID+ginkgolide formulations withastaxanthin and vitamin C. The combined action of the lipid-solublecarotenoid (principally astaxanthin) with vitamin C and one or morecomponents of a Ginkgo biloba extract, in combination with NASAIDsmediates prevention or suppression of disease-associated inflammation.The combination drug therapy described herein utilizes astaxanthinand/or its derivatives; and a gingkolide composition, which containscAMP phosphodiesterase-inhibitory capabilities.

EXAMPLE 4 Compositions of Ginkgolides, Astaxanthin, Plus Vitamin C,Potentiate Anti-Asthmatic Effects of Cetirizine

Cetirizine compounds, e.g., Zyrtec™ (cetirizine hydrochloride), inhibithistamine release by mast cells. Histamine release occurs when mastcells are stimulated, e.g., when antibodies interact with their surfaceH1 receptors (H1R). Selective inhibition of H1R by Zrytec preventsdownstream events, which include intracellular calcium ion release andcalcium uptake and protein kinase C translocation. H1 inhibitioninhibits these effects and also promotes the activation of adenylatecyclase and the resulting accumulation of cAMP.

Components of Ginkgo biloba include terpene antagonists of PAF receptors(PAFR) which synergize with cetirizine and other histamine releaseblockers in reducing the calcium signal (a consequence of PAFRstimulation). Biflavonoid ginkgolides further reduce the effectivedosage of cetirizines by their inhibition of cAMP phosphodiesterase, aneffect which allows augmented accumulation of cAMP.

Astaxanthin also potentiates the effect of cetirizines. Histaminerelease from mast cells is significantly reduced by antioxidants, andastaxanthin further contributes to potentiation of the pharmacologicalactivity of cetirizines.

The compositions described herein are useful to augment the therapeuticactivity of cetirizines such as Zyrtec™, while reducing its effectivedosage. For example, such composition contain a cetirizine compound plusterpene trilactones, such as Gingkolide A, Gingkolide B, Gingkolide C,Gingkolide J, Gingkolide M and bilobalide; or the biflavonoidginkgolides: amentoflavone, bilobetin, sequoiaflavone, ginkgetin andisoginkgetin. The combined action of the lipid-soluble carotenoid (e.g.,astaxanthin) with vitamin C and one or more components of a Ginkgobiloba extract, mediates prevention or suppression of disease-associatedinflammation. The combination drug therapy described herein utilizesastaxanthin and/or its derivatives; and a gingkolide composition, whichcontains cAMP phosphodiesterase-inhibitory as well as antioxidantcapabilities.

Cetirizine compounds such as Zyrtec™ are antihistamines useful ingeneral treatment of allergies, especially seasonal or perennialrhinitis and chronic urticaria. The risk of toxicity associated withsuch compounds is substantially increased in individuals with kidneyimpairment, in particular geriatric patients. The combination drugtherapy regimen (e.g., cetirizine administered with astaxanthin and/or agingkolide), reduces the effective dosage necessary for a beneficialclinical outcome.

The lipid antioxidant astaxanthin and the terpene and biflavonoidcomponents of ginkgo biloba synergize with a cetirizine such as Zyrtec™to reduce H1-mediated histamine release by mast cells and other tissue.The synergistic effect of this combination permits a reduction in theeffective dosage of a cetirizine needed to achieve a desired therapeuticoutcome, thereby reducing adverse side effects of a cetirizine compound.

When cells were cultured in the presence of Ginkgolide B (GB) pluscetirizine, the PMA/Ionomycin-induced expression of the T cellactivation antigen CD25 was suppressed to levels below that mediated byeither GB or Zrytec alone.

Astaxanthin or astaxanthin plus a cetirizine together was administeredto an allergy patient. Astaxanthin alone did not result in alleviationof allergy symptoms. However, the therapeutic effect of the combination(an antioxidant such as astaxanthin and cetirizine) exceeded that ofeither agent alone (as measured by reduction of allergry symptoms suchas itching).

EXAMPLE 5 Suppression of Lymphocyte Activation by Citirazene andAzalestine

Experiments were carried out to determine whether the immunoregulatorycapacity of two commonly-used H1-inhibitory antihistamines: cetirizinedihydrochloride (CTZ/Zyrtec) and azelastine (AZE/Astelin) is potentiatedby the platelet activating factor receptor (PAFR) antagonist and freeradical scavenger Ginkgolide B (GB). For these studies, peripheral bloodmononuclear cells (PBMC) from asthma patients, which were cultured 24hours with either 50 μg/ml PHA or PHA plus selected dosages of each drugwere analyzed by 3-color flow cytometry for expression of CD25+ andHLA-DR+ on CD3+ (T cells). The results shown in Table 3 are reported asstimulation indices (SI) of % CD3+CD25+ cells in cultures treated withPHA alone to % CD3+CD25+ cells in each drug-supplemented culture. Eachdrug was first evaluated independently over a 3-log dose range from10⁻⁸-10⁻⁶ M. Maximal suppression of activation was observed at 10⁻⁸M,where CTZ caused a 29% decrease in SI for CD25+ (p=0.024); and 53% forHLA-DR (p=0.009); with AZE resulting in decreases of 19% for CD25+(p=0.33); and 45% for HLA-DR (p=0.00l); and GB 10⁻⁸ M suppressingHLA-DR+ by 39% (p=0.01). When compared to effects at 10⁻⁸ M, each drugat 10⁻⁷ M showed reduced capacity to independently suppress PHA-mediatedinduction of the two activation antigens. However at this concentration,GB was observed to augment the capacity of CTZ to suppress expression ofCD25+ (p=0.003) and HLA-DR (p=0.004). The suppressive effect of AZE at10⁻⁷ was also potentiated by GB at the same concentration in the case ofCD25+ (p=0.014) and HLA-DR (p=0.000). The data indicated that GBimproved the pharmacological activity of CTZ and AZE at a concentrationof 10⁻⁷M for each of the three components. These data indicate thatGB-augmented antihistamine formulations are useful to alleviate asymptom of asthma-associated inflammation, e.g., abnormal T cellactivation. TABLE 3 Effect of cetirizine/Zyrtec (CTZ), or azalestene(AZE) on induction of CD25+ (CD3+CD25+) and HLA-DR+ (CD3+HLA-DR+) Tlymphocytes in human peripheral blood mononuclear cells (PBMC) P vs N Pvs N Culture SI CD25 Stim subjects SI HLA-DR Stim subjects Unstimulated0.09 ± 0.03 0.000 20 0.450 ± 0.09  0.000 6 Stimulated 1.00 ± 0.00 — 201.00 ± 0.00 — 5 CTZ 10⁻⁸ M 0.71 ± 0.12 0.024 8 0.47 ± 0.13 0.009 5 CTZ10⁻⁷ M 0.88 ± 0.14 0.060 20 0.58 ± 0.11 0.010 5 CTZ 10⁻⁶ M 0.93 ± 0.060.141 20 0.70 ± 0.04 0.001 5 AZE 10⁻⁸ M 0.81 ± 0.08 0.033 8 0.55 ± 0.090.001 5 AZE 10⁻⁷ M 0.88 ± 0.08 0.092 19 0.56 ± 0.10 0.006 5 AZE 10⁻⁶ M1.13 ± 0.15 0.210 17 0.61 ± 0.13 0.018 5 GB 10⁻⁸ M 0.81 ± 0.14 0.105 80.61 ± 0.10 0.010 5 GB 10⁻⁷ M 1.18 ± 0.21 0.196 14 0.62 ± 0.18 0.051 5GB 10⁻⁶ M 0.94 ± 0.12 0.308 15 0.72 ± 0.18 0.100 5 GB 10⁻⁸ M + CTZ 10⁻⁸M 0.79 ± 0.08 0.038 8 0.62 ± 0.13 0.020 5 GB 10⁻⁸ M + CTZ 10⁻⁷ M 0.88 ±0.08 0.096 8 0.75 ± 0.13 0.630 5 GB 10⁻⁸ M + AZE 10⁻⁸ M 0.86 ± 0.090.091 8 0.61 ± 0.04 0.001 5 GB 10⁻⁸ M + AZE 10⁻⁷ M 0.71 ± 0.12 0.024 70.53 ± 0.17 0.035 4 GB 10⁻⁷ M + CTZ 10⁻⁸ M 0.73 ± 0.07 0.002 8 0.64 ±0.10 0.012 5 GB 10⁻⁷ M + CTZ 10⁻⁷ M 0.65 ± 0.09 0.003 8 0.51 ± 0.110.004 5 GB 10⁻⁷ M + AZE 10⁻⁸ M 0.73 ± 0.12 0.033 8 0.62 ± 0.11 0.014 5GB 10⁻⁷ M + AZE 10⁻⁷ M 0.71 ± 0.10 0.014 8 0.50 ± 0.04 0.000 5

EXAMPLE 6 Effects Of Astaxanthin and Ginkgolide B On T LymphocyteActivation

Experiments were carried out to determine whether formulations based onthe platelet activating factor receptor (PAFR) antagonist and freeradical scavenger Ginkgolide B (GB) in combination with the antioxidantcarotenoid astaxanthin (ASX) suppress T cell activation in the same doserange as two commonly-used antihistamines: cetirizine dihydrochloride(CTZ/Zyrtec) and azelastine (AZE/Astelin). Peripheral blood mononuclearcells (PBMC) from asthma patients were cultured 24 hours with either 50μg/ml PHA or PHA plus selected dosages of each drug and analyzed by3-color flow cytometry for expression of CD25+ on CD3+ (T cells).Results are reported as stimulation indices (SI) of % CD3+CD25+ cells incultures treated with PHA alone to % CD3+CD25+ cells in eachdrug-supplemented culture. Formulations which significantly reduced SIof PHA-treated cells ranked in order of increasing magnitude ofsuppression are as follows: ASX 10⁻⁷ M<GB 10⁻⁸M+ASX10⁻⁸M<GB10⁻⁸M<GB10⁻⁷M+ASX 10⁻⁷M<GB 10⁻⁸M+ASX 10⁻⁷M ASX<CTZ 10⁻⁵M<GB10⁻⁶M<GB 10⁻⁷M+ASX 10⁻⁸M<AZE 10⁻⁵M. The data indicate that suppressionof T cell activation below fully-stimulated values by GB, ASX and theircombinations was comparable and for some combinations better than thatmediated by CTZ and AZE.

The studies were carried out as follows.

Patients

Subjects for this study included 12 patients diagnosed with atopicasthma, 7 male and 5 female, ranging in age from 21 to 40 years (mean28±1.8 years). Disease duration ranged from 2 to 12 years. Atopy wasdefined on the basis of one or more positive skin prick tests to a rangeof 20 allergens. None of the patients had received systemic therapy forat least 6 weeks prior to blood collection. The mean serum IgE was 335(170-480) IU/ml.

Cell Cultures

Venous blood for each subject was collected in polyethylene tubescontaining EDTA during a one hour morning time interval. PBMC wereseparated by Ficoll-paque (Pharmacia, Uppsala, Sweden) density gradientcentrifugation. Cells were washed and suspended in RPMI 1640 medium(Gibco BRL, Gaithersburg, Md.) at density of 1×10⁶ cells/ml. PBMC werestimulated with 50 μg/ml Phytohemaglutinin (PHA) (SigmaImmunopharmaceuticals, St. Lous Mo.), or PHA plus 10⁻⁸-10⁻⁵ Mastaxanthin (Natural Alternatives International (NAI) Inc., San MarcosCalif.); or ginkgolide B 10⁻⁸-10⁻⁶ M (NAI San Marcos Calif.); orselected combinations of ASX plus GB. Comparison of ASX and GB effectson T cell activation were made with two other pharmacological agentswith anti-asthmatic properties by treating cells with 10⁻⁷-10⁻⁴ Mcetirizine dihydrochloride (Pfizer Pharmaceuticals, Norwich Conn.); or10⁻⁷-10⁻⁴ M azalestine hydrochloride (Wallace Pharmaceuticals, SomersetN.J.), followed by evaluation of cultures for the same biologicalendpoints as ASX/GB-treated cells. Each reagent was added at the outsetof a 24 hours culture period, followed by harvest of cellular fractionfor immunophenotyping studies.

Flow Cytometric Analysis

Cells harvested from cultures by centrifugation were incubated for 30min at 4° C. with 10 μl each of flourescein-isothiocyanate (FITC)-CD3and phycoerythrin (RD1)-CD25 conjugated monoclonal antibodies (mAb)(Dakopatts, A/S, Glostrup, Denmark), followed by fixation withparaformaldehyde. Two-color Flow cytometry was conducted using a CoulterEpics XL automated flow cytometer (Coulter Scientific, Hialeah, Fla.,USA). Isotypic controls for the monoclonal antibodies (mAb) used todetect antigens of interest were established for each cell preparation.Positive analysis regions for cells expressing specific surface markerswere set against controls and specific binding of fluorophore-conjugatedmAb was analyzed by cytofluorography according to standard methodsrecommended by the manufacturer. Lymphocyte subpopulations wereidentified by position on forward and side scatter plots and live-gated.Expression of each antigen was reported as percentage cells positive fora particular T cell subpopulation defined by expression of CD3 (Tlymphocyte marker) plus CD25, plus or minus standard error.

Statistical Analysis

Statistical analysis was performed using an independent t-test. Allstatistical analyses were performed using the SPSS for Windowsstatistical package (Norusis/SPSS, Inc.). A value of p<0.05 wasconsidered statistically significant

T Lymphocyte Activation

Culture of PBMC for 24 hours with 50 μg/ml of the immunostimulatorylectin PHA resulted in significant activation of T lymphocytes, measuredas increased percentage of CD3+CD25+ cells versus unstimulated cultures(Table 3). The capacity of formulations evaluated in this study tosuppress immune activation was measured as a stimulation index (SI),defined as the ratio of CD3+CD25+ cells in each test condition toCD3+CD25+ in cultures treated with PHA alone. Assigning fully-stimulatedcultures an SI value of 1.00, we observed that 9 of the 26 candidateformulations resulted in significant (p<0.05) reduction in SI (Table 1).

Effects of Astaxanthin and GB on T Lymphocyte Activation

As shown in FIG. 1, stimulation indices for PHA-treated cells weresuppressed significantly by astaxanthin at a concentration of 10⁻⁷ M(SI=0.89±0.06, p<0.034). Ginkgolide B significantly reduced SI ofPHA-stimulated cells at dosages of 10⁻⁶M (SI=0.77±0.12, p=0.048); and10⁻⁸M (SI=0.86±0.07, p=0.05) (FIG. 2). Combinations of these agents alsosignificantly suppressed immune activation. These formulations included10⁻⁷M GB in combination with 10⁻⁷M ASX (SI=0.86±0.06, p=0.037); 10⁻⁷MGB+10⁻⁸M ASX (SI=0.77±0.05, p=0.006); ₁₀ ⁻⁸ M GB+10⁻⁷ M ASX(SI=0.85±0.05, p=0.015); and 10⁻⁸M GB+10⁻⁸M ASX (SI=0.87±0.06, p=0.040)(FIG. 3); and cells stimulated with a combination of 10−8M ASX plus10⁻⁷M ginkgolide B, which suppressed induction of CD3+CD25+ cells to anSI of 0.77±0.05, significantly below the suppression mediated by 10⁻⁸MASX alone acting on PHA-stimulated cultures (p=0.051) (FIGS. 1 and 3).Nevertheless treatment of cells with 10⁻⁷M GB+10⁻⁸M ASX failed tosignificantly suppress activation below 10⁻⁷M GB alone acting onPHA-treated cells (p=0.373) (FIGS. 2 and 3).

Effects of Cetirizine and Azelastine on T Lymphocyte Activation

Two commonly-used anti asthmatic compounds, cetirizine dihydrochloride(Zyrtec, CTZ) and azelastine HCl (Astelin, AZE) were evaluated under thesame conditions as ASX and GB for their ability to suppress T cellactivation. Cells treated with PHA exhibited significant reduction ininduction of CD3+CD25+ cells at a concentration of 10⁻⁵M for bothCTZ(SI=0.78±0.11, p=0.05) (FIG. 4A); and AZE (SI=0.76±0.12, p=0.034)(FIG. 4B).

Combination Drug Therapy for Inhibition of T Cell Activation in AsthmaSubjects

Asthma is associated with elevated expression in bronchoalveolar tissueof Th2 cytokines (IL-3, IL-5, and GM-CSF), which in turn upregulateeosinophil recruitment, activation, proliferation and differentiation,promoting tissue injury and fibrosis via an increased production of avariety of toxic metabolites. Histamine release blockers such asazalestine and cetirizine which treat the disease downstream from theunderlying pathogenic T lymphocyte activity have been successful inpartially alleviating its symptoms, but are often not as effective asagents, which directly suppress abnormal T cell activation. Neverthelesssince cellular signaling pathways which promote tissue damage in asthma,exert positive feedback and increase T cell activation, drugs whichinhibit release or activity of inflammatory metabolites are alsoexpected to exhibit immunosuppressive properties. Indeed, the H1receptor antagonist terfenadine is observed to inhibit proliferation andexpression of IL-4 and IL-5 production by anti-CD3/-CD28 andPMA-activated human T cells in vitro. Since both of these Th2 cytokinesare implicated as major factors in asthma pathogenesis, therapeuticeffects of this drug are likely mediated at least in part by suppressionof T cell activity.

Ginkgolide B and astaxanthin with azalestine and cetirizine were testedfor the ability to suppress T cell activation in PHA-stimulated culturesof human PBMC taken from asthma patients. These experiments weredesigned with the recognition that suppression of T lymphocyteactivation is not the primary mechanism by which each compound mediatesits therapeutic effects. However, since T cell activity is a criticalcomponent of the cascade of signaling events resulting in the symptomsof asthma, T cell suppression represents a useful index to gauge therelative effectiveness of the pharmacological agents tested. Table 3shows the effect of each stimulation condition on cells with respect totheir ability to inhibit PHA-induced upregulation of the IL-2 receptor(CD25) on CD3+ cells (an index of T cell activation). When astaxanthinalone was added to PHA-treated cultures, significant suppression of Tlymphocyte activation occurred at a concentration of 10⁻⁷M (FIG. 1);whereas SI values significantly lower than 1.00 (fully stimulated) wereobserved over a 3 log dose range of ginkgolide B, with SI valuessignificantly less than 1.00 observed at GB concentrations of 10⁻⁸ M and10⁻⁶ M (FIG. 2). When combinations of ASX and GB were evaluated fortheir capacity to suppress T cell activation, four combinations of thecompounds were observed to result in significant reduction inPHA-mediated induction of CD3+CD25+ cells; with an optimal combinationoccurring at a concentration of 10⁻⁸ M ASX plus 10⁻⁷ M GB (FIG. 3).Mechanisms contributing to suppression of T cell activation by ASX, GBand their combination are likely a consequence of the major biochemicalproperties of each compound acting together. Reactive oxygen species(ROS) are substantially upregulated by T lymphocytes during PHA-mediatedactivation; moreover blocking this enhancement with antioxidants altersthe activation process.

Although previous studies of cetirizine suggest that it has nosignificant effect on T cells, the data described herein indicate thatat an optimal concentration of 10⁻⁵ M, it will suppress at least thoseaspects of T cell activation involving expression of CD25 (FIG. 4A).Cetirizine also displays an ability to downregulate aspects of T cellactivation related to chemotaxis. The present results indicate thatastaxanthin and ginkgolide B act in concert to mediate antiasthmaticeffects as well or better than currently-used medications. Compositionscontaining the combination of compounds described herein reduceinflammation (e.g., by inhibiting T cell activation) with little or noneof the side effects associated with conventional anti-inflammatorymedicaments. When these compositions are administered in conjunctionwith conventional anti-inflammatory drugs, less of the conventional drugis required to achieve the same or similar therapeutic benefit, therebyreducing undesirable side effects associated with the conventional drug.

EXAMPLE 7 Compositions Containing Ginkolide, Astaxanthin and Vitamin CSuppress Allergen Induced Asthma

The effect combinations of vitamin C, astaxanthin, and ginkolide onasthma-associated disease parameters ovalbumin(OA)-induced asthma inguinea pigs was evaluated. Twenty-four hours following OA challenge,animals are sacrificed and numbers of inflammatory cells (eosinophils,neutrophils, macrophages) are measured in bronchoalveolar lavage (BAL)fluid; and cAMP and cGMP concentrations in the lung tissue. Theexperiments were carried out as follows.

Asthmatic Model (Sensitization Procedure):

Male Hartley guinea pigs (250-350 g) were sensitized by intramuscularinjections of 0.35 ml of a 5% (W/V) ovalbumin (OA)/saline solution intoeach thigh on days 1 and 4. Guinea pigs were ready for asthmaticchallenge after 25 days of ovalbumin injection. Asthmatic challenge wascarried out with ovalbumin aerosol, and bronchoalveolar lavage (BAL) wasdone 24 hours later. The numbers of eosinophils, neutrophils, andmacrophages in the BAL fluid were counted.

Measurement of cAMP and cGMP:

Biopsies from lung tissues, cAMP and cGMP were measured usingcommercially available radioimmunoassay kits (Amersham). Immediatelyafter sampling, lung biopsies were frozen by means of a Wollenbergerclamp prechilled in liquid nitrogen. Samples were powdered with a pestleand mortar in liquid nitrogen and trichloro acetic acid (TCA) was addedto the powdered frozen samples (10 ml to every mg of tissue). Sampleswere further homogenized in the frozen TCA in the braying mortar andthen centrifuged at 14,000×g for 10 min at 4° C. The supernatants wereextracted 6 times in water-saturated diethylether, evaporated andassayed for cGMP by radioimmunoassay using liquid scintillation counter(Packard, Tri-Carb 2100TR).

Experimental Time Course and Measurement of Cells in BAL Fluid:

Twenty-four hours after the aerosol OA challenge, guinea pigs werekilled by cervical dislocation and exsanguinated by severing theaxillary arteries. Lungs were lavaged with 50 ml of DulBecco'sphosphate-buffered saline (aliquots of 10 ml), which were aspiratedafter gentle chest massage. BAL fluid was centrifuged at 2200 rpm(1100×g) for 10 min, supernatant was aspirated, and pellets wereresuspended in 5 ml 0.25% NaCl to lyse residual erythrocytes. Thisdispersion was centrifuged at 2200 rpm (1100×g) for 10 min, supernatantwas aspirated, and pellets were resuspended 5 ml 0.9% NaCl. Total cellcounts were done by hemocytometry using trypan blue stain. Slides wereprepared on a Shandon Cytospin 2 (Pittsburgh, Pa.) at 300 rpm for 5 min,fixed and stained. Differential cell counts were done using standardmorphologic criteria to classify cells as eosinophils, neutrophils, ormacrophages, and the results were expressed in cell numbers.

Dosage Effects of Vitamin C, AX, and GB Administered Singularly

The dose-responsive effect of AX, EGb761, and vitamin C administeredindependently for suppression of asthma-associated parameters inallergen-induced, asthmatic guinea pigs was determined. Ibuprophen (IB)was used as the positive control. The results are shown in Tables 4-7.Concentration of inflammatory cells in bronchoalveolar lavage (BAL)fluid and levels of cAMP and cGMP in lung tissue were measured in guineapigs 24 hours following challenge with OV. Data is reported as mean±SDof measurements taken in 6 animals per dosage cohort. *p<0.05 comparedto corresponding values of OV antigen-challenged, drug-free controlgroup.

As shown in Tables 1-3, these components used separately (10 mg/kg AX,200 mg/kg of vit C, and 10 mg/kg of GB) failed to protect the OV-inducedasthma. TABLE 4 Astaxanthin (AX)-induced change in asthma-associatedparameters Control, Asthma- Control Cohort size: free in OV AX AX AX AxAx n = 6 group asthma 5 mg/kg 10 mg/kg 30 mg/kg 100 mg/kg 200 mg/kgEosinophils 1.4 ± 0.2 5.4 ± 0.8 5.0 ± 0.7 4.6 ± 0.6 3.1 ± 0.5* 2.4 ±0.6* 2.3 ± 0.5* (cell × 10⁶/ animal). Neutrophils 1.1 ± 0.3 5.8 ± 0.75.4 ± 0.8 5.1 ± 0.6 3.3 ± 0.8* 2.4 ± 0.5* 2.0 ± 0.4* (cell × 10⁵/animal) Macrophages 1.7 ± 0.5 9.9 ± 1.1 9.6 ± 1.0 8.0 ± 0.8 3.7 ± 0.7*2.70 ± 0.7* 2.6 ± 0.5* (cell × 10⁶/ animal) cAMP pmol 10.9 ± 0.98  6.7 ±0.58 6.9 ± 0.5 8.1 ± 0.8 9.6 ± 0.8* 12.6 ± 0.8* 12.1 ± 0.5*  (mgprotein)⁻¹ cGMP pmol  2.6 ± 0.26  1.1 ± 0.17 1.2 ± 0.1 1.4 ± 0.1 1.8 ±0.3*  2.8 ± 0.3* 2.7 ± 0.2* (mg protein)⁻¹

TABLE 5 Ginkgo biloba (EGb761)-induced, change in asthma-associatedparameters Control, Asthma- Control Cohort size: free in OV EGb761EGb761 EGb761 EGb761 n = 6 group asthma 5 mg/kg 10 mg/kg 30 mg/kg 100mg/kg Eosinophils (cell × 10⁶/ 1.4 ± 0.2 5.4 ± 0.8 5.1 ± 0.9 5.0 ± 0.83.5 ± 0.8* 3.1 ± 0.5* animal). Neutrophils (cell × 10⁵/ 1.1 ± 0.3 5.8 ±0.7 5.6 ± 0.6 5.4 ± 0.6 3.5 ± 0.7* 3.3 ± 0.6* animal) Macrophages 1.7 ±0.5 9.9 ± 1.1 9.8 ± 1.1 9.4 ± 1.1 4.9 ± 1.5* 4.2 ± 0.6* (cell × 10⁶/animal) cAMP pmol (mg 10.9 ± 0.98  6.7 ± 0.58 6.9 ± 0.6 8.0 ± 0.9 9.8 ±0.7* 12.0 ± 1.3*  protein)⁻¹ cGMP pmol (mg  2.6 ± 0.26  1.1 ± 0.17 1.1 ±0.2 1.1 ± 0.2 1.5 ± 0.2* 1.8 ± 0.3* protein)⁻¹

TABLE 6 Vitamin C-induced change in asthma-associated parametersControl, Asthma- Control Cohort size: free in OV Vitamin C Vitamin CVitamin C Vitamin C n = 6 group asthma 50 mg/kg 100 mg/kg 200 mg/kg 400mg/kg Eosinophils (cell × 10⁶/ 1.4 ± 0.2 5.4 ± 0.8 5.5 ± 0.5 5.5 ± 0.84.8 ± 1.1 3.5 ± 0.9* animal). Neutrophils (cell × 10⁵/ 1.1 ± 0.3 5.8 ±0.7 5.4 ± 0.6 5.6 ± 0.8 5.0 ± 0.8 4.7 ± 0.9  animal) Macrophages 1.7 ±0.5 9.9 ± 1.1 9.5 ± 0.9 9.8 ± 0.9 9.2 ± 0.8 7.2 ± 1.5* (cell × 10⁶/animal) cAMP pmol (mg 10.9 ± 0.98  6.7 ± 0.58 6.8 ± 0.5 7.1 ± 0.8 7.9 ±0.9 7.9 ± 0.8  protein)⁻¹ cGMP pmol (mg 2.6 ± 0.26  1.1 ± 0.17 1.1 ± 0.21.0 ± 0.2 1.2 ± 0.2 1.5 ± 0.2* protein)⁻¹

TABLE 7 Ibuprophen (IB)-induced change in asthma-associated parametersControl, Asthma- Control Cohort size: free in OV IB IB IB IB n = 6 groupasthma 10 mg/kg 100 mg/kg 500 mg/kg 1000 mg/kg Eosinophils (cell × 10⁶/1.4 ± 0.2 5.4 ± 0.8 5.4 ± 0.8 4.7 ± 0.6 3.0 ± 0.8* 2.9 ± 0.5* animal).Neutrophils (cell × 10⁵/ 1.1 ± 0.3 5.8 ± 0.7 5.7 ± 0.7 5.0 ± 0.7 3.1 ±0.9* 3.1 ± 0.5* animal) Macrophages 1.7 ± 0.5 9.9 ± 1.1 9.5 ± 0.8 9.0 ±1.3 4.7 ± 1.3* 4.3 ± 0.9* (cell × 10⁶/ animal) cAMP pmol (mg 10.9 ± 0.98 6.7 ± 0.58 7.1 ± 0.6 7.3 ± 0.9 8.2 ± 0.8* 8.1 ± 0.9* protein)⁻¹ cGMPpmol (mg  2.6 ± 0.26  1.1 ± 0.17 1.1 ± 0.2 1.1 ± 0.2 1.4 ± 0.2  1.6 ±0.2* protein)⁻¹Determine the Optimal Doses of the Combination of AX EGb761, and VitaminC for the Prevention of Asthma in Guinea Pigs Induced by Allergen.

The dose-responsive effect of AX, EGb761, and vitamin C administered incombination for suppression of asthma-associated parameters inallergen-induced, asthmatic guinea pigs was determined. The combinationof these three components was given each day for each guinea pig as a“cocktail”. The results are shown in Table 8. Concentration ofinflammatory cells in bronchoalveolar lavage (BAL) fluid and levels ofcAMP and cGMP in lung tissue were measured in guinea pigs 24 hoursfollowing challenge with OV. Data is reported as mean±SD of measurementstaken in 6 animals per dosage cohort. *p<0.05 compared to correspondingvalues of OV antigen-challenged, drug-free control group.

The combination of AX (10 mg/kg), vit C (200 mg/kg), and EGb761 (10mg/kg) produced a significant protection against OA-induced asthma,while these concentrations (using separately of each drug) of AX (10mg/kg), vit C (200 mg/kg), and GB (10 mg/kg) failed to reduce theseverity of asthma. These results demonstrate that combinations ofastaxanthin, Ginkgo biloba leaf extract and vitamin C exhibit potentanti-inflammatory potential which is equal or superior to Ibuprophen(IB—Table 7). These formulation are superior to IB as they do not causethe gastrointestinal problems typically associates with IB. TABLE 8Change in asthma-associated parameters induced by formulations composedof astaxanthin, vitamin C and Ginkgo biloba leaf extract (EGb761). 10mg/kg 30 mg/kg Control, Control 5 mg/kg AX + 50 mg/kg AX + 200 mg/kgAX + 400 mg/kg Asthma- in Vit Vit Vit Cohort size: free OV C + 5 mg/kgC + 10 mg/kg C + 30 mg/kg n = 6 group asthma EGb761 EGb761 EGb761Eosinophils 1.52 ± 0.15 5.63 ± 0.90 5.07 ± 0.63 3.44 ± 0.84* 2.95 ±0.62* (cell × 10⁶/ animal). Neutrophils 1.00 ± 0.31 5.92 ± 0.66 5.27 ±0.74 3.65 ± 0.65* 2.60 ± 0.69* (cell × 10⁵/ animal) Macrophages 1.52 ±0.40 10.18 ± 0.88  9.40 ± 0.96 5.78 ± 1.18* 2.98 ± 0.71* (cell × 10⁶/animal) cAMP pmol 10.77 ± 0.74  6.75 ± 0.81 7.17 ± 0.61 9.28 ± 0.94*13.00 ± 0.76*  (mg protein)⁻¹ cGMP pmol 2.72 ± 0.12 1.02 ± 0.15 1.17 ±0.19 2.09 ± 0.48* 2.84 ± 0.28* (mg protein)⁻¹

These data demonstrate the effect of the combination of Ginkgo biloba(EGb761) astaxanthin (AX) and vitamin C to suppress features of asthmapathogenesis. Specifically, this formulation suppressed inflammatoryprocesses including decreased inflammatory cell infiltration and cAMPand cGMP concentration in tissue. These data support the usefulness ofthe formulation described herein to alleviate symptoms of asthma andother inflammatory disorders that are associated with similarinflammatory processes. For example dry eye, an aqueous tear-deficientdry eye syndrome which results disruption of the ocular surface-lacrimalgland homeostatic cycle. Dry eye is characterized by dry inflammation ofthe lacrimal gland, and presence of a dense infiltrate of inflammatorycells in and around the tear duct causing high localized expression ofpro-inflammatory cytokines.

EXAMPLE 8 Effect of Asataxanthin (ASX), Ginkgolide B and theirCombinations on PHA-Mediated Induction of CD3+CD25+ or CD3+HLA-DR+Lymphocytes in Human Peripheral Blood Mononuclear Cells (PBMC)

Cells from 3-12 asthma patients were cultured 24 hours with 50 μg/mlPHA. PBMC evaluated by 2-color flow cytometry were gated for CD3+ andanalyzed for CD3+CD25+ or CD3+HLA-DR+ as a percentage of the CD3+population. Results are reported as stimulation indices (SI) calculatedas the ratio of % CD3+CD25+ cells or % CD3+HLA-DR+ cells infully-stimulated cultures to % CD3+CD25+ or % CD3+HLA-DR+ respectivelyin cells treated with PHA plus ASX, GB, or combinations thereof. Boldedentry demonstrates effect of GB+ASX, more effective than either at sameconcentration in culture. The results are shown in Table 9 below. TABLE9 CD3+HLA- CD3+CD25+ P versus DR+ P versus Culture StimulationStimulated N Stimulation Stimulated N condition Index cultures SubjectsIndex cultures Subjects Unstim 0.05 ± 0.01 0.000 12 0.40 ± 0.08 0.008 4Stim  1.0 ± 0.00 11 1.00 ± 0.00 4 ASX 10⁻⁸ M 0.95 ± 0.05 0.174 8 0.68 ±0.16 0.067 4 ASX 10⁻⁷ M 0.90 ± 0.05 0.032 10 0.71 ± 0.23 0.164 3 ASX10⁻⁶ M 0.96 ± 0.10 0.350 10 0.83 ± 0.23 0.267 3 ASX 10⁻⁵ M 0.95 ± 0.060.205 11 0.79 ± 0.19 0.193 3 ASX 10⁻⁴ M 0.49 ± 0.09 0.000 11 0.85 ± 0.180.191 3 GB 10⁻⁸ M 0.90 ± 0.07 0.052 9 0.73 ± 0.12 0.077 3 GB 10⁻⁷ M 0.92± 0.08 0.161 10 0.78 ± 0.20 0.194 3 GB 10⁻⁶ M 0.83 ± 1.0 0.060 10 0.66 ±0.06 0.001 3 GB 10⁻⁸ M + ASX 0.90 ± 0.06 0.057 7 0.74 ± 0.15 0.116 310⁻⁸ M GB 10⁻⁸ M + ASX 0.90 ± 0.05 0.056 8 0.91 ± 0.17 0.317 4 10⁻⁷ M GB10⁻⁸ M + ASX 0.90 ± 0.06 0.072 9 0.79 ± 1.0 0.055 4 10⁻⁶ M GB 10⁻⁷ M +ASX 0.82 ± 0.04 0.004 6 0.82 ± 0.11 0.071 4 10⁻⁸ M GB 10 ⁻⁷ M + ASX 0.88± 0.05 0.018 10 0.78 ± 0.09 0.048 4 10 ⁻⁷ M GB 10⁻⁷ M + ASX 0.94 ± 0.060.188 10 0.61 ± 0.05 0.002 4 10⁻⁶ M GB 10⁻⁶ M + ASX 0.90 ± 0.04 0.031 60.81 ± 0.13 0.118 4 10⁻⁸ M GB 10⁻⁶ M + ASX 0.87 ± 0.08 0.074 8 0.73 ±0.16 0.094 4 10⁻⁷ M GB 10⁻⁶ M + ASX 0.93 ± 0.07 0.185 7 0.80 ± 0.170.174 3 10⁻⁶ MOther embodiments are within the following claims.

1. A method of reducing inflammation of an ocular tissue comprisingcontacting said tissue with a composition comprising a carotenoid and apolyphenol.
 2. The method of claim 1, wherein said composition furthercomprises a glutathione precursor.
 3. The method of claim 1, whereinsaid composition further comprises a vitamin anti-oxidant.
 4. The methodof claim 1, wherein said composition further comprises an alpha lipoicacid.
 5. The method of claim 1, further comprising contacting saidtissue with an omega-3 fatty acid.
 6. The method of claim 1, furthercomprising contacting said tissue with an omega-6 fatty acid.
 7. Themethod of claim 5, wherein said omega-3 fatty acid is selected from thegroup consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid(DHA), and alpha linolenic acid (ALA).
 8. The method of claim 1, whereinsaid carotenoid is a mixed carotenoid compound, a purified astaxanthin,or a purified zeaxanthin.
 9. The method of claim 1, wherein saidpolyphenol is curcuma longa root powder, green tea, grape seed extract,a cinnamon flavonoid, or a citrus bioflavonoid.
 10. The method of claim1, wherein said polyphenol is a cox-2 inhibitor.
 11. The method of claim10, wherein said cox-2 inhibitor is a quercetin, a bilberry extract, ahops PE, blueberry powder or tart cherry powder.
 12. The method of claim2, wherein said glutathione precursor is taurine or N-acetyl-L-cysteine.13. The method of claim 3, wherein said vitamin anti-oxidant is VitaminA, Vitamin B, Vitamin C, Vitamin D or Vitamin E.
 14. The method of claim1, wherein said composition further comprises a trace mineral.
 15. Themethod of claim 1, wherein said composition further comprisesL-carnitine.
 16. The method of claim 1, wherein said ocular tissue issclera tissue, iris tissue, cornea tissue, pupil tissue, lens tissue,conjuctiva tissue, vitreous tissue, choroids tissue, macula tissue orretina tissue
 17. A method of alleviating a symptom of dry eye ormacular degeneration, comprising administering to a subject sufferingfrom or at risk of developing dry eye or macular degeneration acomposition comprising a carotinoid and a polyphenol.
 18. The method ofclaim 17, wherein said composition further comprises a glutathioneprecursor.
 19. The method of claim 17, wherein said composition furthercomprises a vitamin anti-oxidant.
 20. The method of claim 17, whereinsaid composition further comprises an alpha lipoic acid.
 21. The methodof claim 17, further administering said subject an omega-3 fatty acid.22. The method of claim 21, wherein said omega-3 fatty acid is selectedfrom the group comprising EPA, DHA, and ALA.
 23. The method of claim 17,wherein said composition further comprises an omega-6 fatty acid. 24.The method of claim 17, wherein said carotenoid is a mixed carotenoidcompound, a purified astaxanthin or a purified zeaxanthin.
 25. Themethod of claim 17, wherein said polyphenol is curcuma longa rootpowder, green tea, grape seed extract, cinnamon flavonoid, or a citrusbioflavonoid.
 26. The method of claim 17, wherein said polyphenol is acox-2 inhibitor.
 27. The method of claim 26, wherein said cox-2inhibitor is a quercetin, a bilberry extract, a hops PE, blueberrypowder or tart cherry powder.
 28. The method of claim 18, wherein saidglutathione precursor is taurine or N-acetyl-L-cysteine.
 29. The methodof claim 19, wherein said vitamin anti-oxidant Vitamin A, Vitamin B,Vitamin C, Vitamin D or Vitamin E.
 30. The method of claim 17, whereinsaid composition further comprises a trace mineral.
 31. The method ofclaim 17, wherein said composition is administered systemically.
 32. Themethod of claim 17, wherein said composition is administered locally.33. The method of claim 32, wherein said composition is administered bydirectly contacting an ocular tissue.
 34. The method of claim 17,further comprising contacting said tissue with L-carnitine.
 35. Acomposition comprising a carotenoid and a polyphenol, wherein saidcarotenoid and said polyphenol are present in amounts to produce asynergistic anti-inflammatory effect.
 36. The composition of claim 35,wherein said composition further comprises a glutathione precursor. 37.The composition of claim 35, wherein said composition further comprisesa vitamin anti-oxidant.
 38. The composition of claim 35, wherein saidcomposition further comprises an alpha lipoic acid.
 39. The compositionof claim 35, wherein said carotenoid is a mixed carotenoid compound, apurified astaxanthin or a purified zeaxanthin.
 40. The composition ofclaim 35, wherein said polyphenol is curcuma longa root powder, greentea, grape seed extract, cinnamon flavonoid, or a citrus bioflavonoid.41. The composition of claim 35, wherein said polyphenol is a cox-2inhibitor.
 42. The composition of claim 41, wherein said cox-2 inhibitoris a quercetin, a bilberry extract, a hops PE, blueberry powder or tartcherry powder.
 43. The composition of claim 36, wherein said glutathioneprecursor is taurine or N-acetyl-L-cysteine.
 44. The composition ofclaim 37, wherein said vitamin anti-oxidant is Vitamin A, Vitamin B,Vitamin C, Vitamin D or Vitamin E.
 45. The composition of claim 35,wherein said composition further comprises a trace minerals.
 46. Thecomposition of claim 35, wherein said composition further comprisesL-carnitine.